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Published March 22, 2023 | Submitted
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MicroED structure of a protoglobin reactive carbene intermediate


Microcrystal electron diffraction (MicroED) is an emerging technique which has shown great potential for describing new chemical and biological molecular structures. [1] Several important structures of small molecules, natural products and peptides have been determined usingab initiomethods. [2] However, only a couple of novel protein structures have thus far been derived by MicroED. [3, 4] Taking advantage of recent technological advances including higher acceleration voltage and using a low-noise detector in counting mode, we have determined the first structure of anAeropyrum pernixprotoglobin (ApePgb) variant by MicroED using an AlphaFold2 model for phasing. The structure revealed that mutations introduced during directed evolution enhance carbene transfer activity by reorienting an alphahelix ofApePgb into a dynamic loop making the catalytic active site more readily accessible. After exposing the tiny crystals to substrate, we also trapped the reactive iron-carbenoid intermediate involved in this engineeredApePgb's new-to-nature activity, a challenging carbene transfer from a diazirine via a putative metallo-carbene. The bound structure discloses how an enlarged active site pocket stabilizes the carbene bound to the heme iron and, presumably, the transition state for formation of this key intermediate. This work demonstrates that improved MicroED technology and the advancement in protein structure prediction now enables investigation of structures that were previously beyond reach.

Additional Information

The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. E.D. thanks The Wenner-Gren Foundations for their support through the Wenner-Gren Postdoctoral Fellowship. This study was supported by the National Institutes of Health P41GM136508. The Gonen laboratory is supported by funds from the Howard Hughes Medical Institute. N.J.P. thanks Merck and the Helen Hay Whitney Foundation for their support through the Merck-Helen Hay Whitney Foundation Postdoctoral Scholarship. This publication is based on work supported by the United States Army Research Office under Contract W911NF-19-0026 for the Institute for Collaborative Biotechnologies and the G. Harold and Leila Y. Mathers Charitable Foundation. Author contributions. N.P. conducted protein expression and crystallization experiments. E.D. prepared the samples and conducted MicroED data collection. E.D. and J.U. analyzed the data and solved the structures. E.D., J.U., N.P, F.A, and T.G. took part in preparation of the manuscript. The authors have declared no competing interest.

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August 20, 2023
August 20, 2023